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Novel approaches in the detection and characterisation of circulating and micrometastatic tumour cells in epithelial malignanciesTheocharous, Panteli January 2001 (has links)
No description available.
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Isolation and characterization of porcine monocyte-derived mesenchymal cellsCaballero Vidal, Cesar Guillermo January 1900 (has links)
Doctor of Philosophy / Department of Animal Sciences and Industry / Duane L. Davis / Monocytes are leukocytes in peripheral blood that differentiate into macrophages in the context of the inflammatory response. Leukocytes are easy to isolate from a blood sample by inexpensive standardized methods, such as the Ficoll-based density gradient. We have found that monocytes isolated from peripheral blood of pigs and grown using simple procedures produce large numbers of mesenchymal cells that exhibit differentiation into mesodermal lineages in vitro.
Peripheral blood samples were obtained from 2, 4, and 6 months old male pigs. The cells were isolated by a Ficoll-based density gradient and cultured in 20% FBS in DMEM media, on uncoated tissue culture vessels. All isolates exhibited mesenchymal morphology and continued to expand at least to passage 7. The expansionary potential was greatest for the cells obtained from the 2 mo. old pigs. We isolated similar cells from porcine fetal livers (gestation day 60), at which time hematopoiesis is occurring in the liver. Therefore, these cells are present from at least mid-gestation through 6 months, the approximate age of puberty in pigs.
In regards to immune-phenotype, the cells are strongly positive for the leukocyte maker CD 14 and SLA-DR-II. Approximately 50% of the cells are positive for CD 45, and they are negative for CD 105, CD 31, and CD 90. The monocyte-derived cells express mRNAs for TLR-3,4,5, 7, and 9. They also express the pluripotency-associated gene Nanog but only weakly express Sox-2 and Oct-4. In vitro the cells are capable of differentiation into adipogenic, osteogenic, and chondrogenic lineages. They also exhibit phagocytosis as measured by in vitro assay. We tested their ability to support the porcine reproductive and respiratory virus in vitro but they were not supportive using standard techniques. Initial attempts have also failed to support myogenic differentiation.
The cells isolated in this study represent a novel subset of monocytes with characteristics overlapping those of mesenchymal stem cells. Swine are physiologically similar to humans and further work is needed to characterize these cells for regenerative medicine applications.
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Apport du transcriptome des cellules mononucléées sanguines à l’étude de cas familiaux et sporadiques atteints de la maladie de Parkinson / Contribution of the transcriptome of peripheral blood mononuclear cells to the study of familial and sporadic cases with Parkinson's diseaseMutez, Eugénie 30 November 2011 (has links)
La maladie de Parkinson (MP) est caractérisée par la mort des neurones dopaminergiques de la substance noire et la présence de corps de Lewy. Son diagnostic reste sujet à des erreurs notamment aux stades précoces. Les cellules mononucléées sanguines périphériques (PBMC) jouent un rôle dans la cascade délétère et sont le reflet d’événements associés à la MP. Même si elles ne représentent qu’un faible pourcentage, les formes génétiquement déterminées permettent d’identifier des sujets à un stade précoce. Nous avons émis l’hypothèse que les PBMC pouvaient constituer un modèle d’étude reflétant certains mécanismes de la dégénérescence du vivant du patient. Nous avons réalisé des études du transcriptome chez différents groupes de sujets malades ou porteurs de mutations pour y déceler les gènes et voies de signalisation cellulaire dérégulés. Nous avons d’abord étudié le profil d’expression génique de sujets porteurs de la mutation G2019S de LRRK2. L’analyse des puces a permis d’identifier des perturbations de voies impliquées dans la MP comme l’oxydation mitochondriale, l’inflammation et la guidance axonale. Des altérations de la voie des MAPK, du cytosquelette d’actine et du transport vésiculaire ont été notées. La liste des gènes dérégulés permet de séparer les individus selon leur statut génétique. La mutation LRRK2 est associée à un profil d’expression génique dès les stades précoces identifiable dans les PBMC. Nous nous sommes ensuite intéressés à une autre forme de MP avec duplication de SNCA. Nous avons caractérisé la relation entre le génotype et le phénotype clinique des sujets de cette famille. La duplication s’étend sur 4,928 Mb, comporte 31 gènes et résulte d’une recombinaison homologue non allélique. L’analyse de l’expression des gènes présents dans la duplication dans les PBMC d’un sujet à un stade pauci-symptomatique a montré une surexpression de SNCA. Nous avons comparé nos analyses chez les porteurs des mutations LRRK2 et SNCA et chez des parkinsoniens sporadiques. Nos analyses montrent que les sujets LRRK2 et les sujets sporadiques présentent des dérégulations communes de voies de signalisation. En revanche, les voies dérégulées chez le sujet dupliqué reflètent la pathogénie de SNCA comme l’autophagie et les voies lysosomales. Nous nous sommes intéressés à l’expression des 4 isoformes de SNCA dans les PBMC de ces 3 groupes d’individus. Les patients sporadiques et LRRK2 montrent une diminution de l’expression des 4 isoformes de SNCA dans leur PBMC. Chez le sujet dupliqué, on observe uniquement une surexpression de l’isoforme 112. Nous avons ensuite identifié les voies moléculaires associée / Parkinson's disease (PD) is prone to misdiagnosis particularly in the early stages. A better understanding of the deleterious mechanisms is essential to identify therapeutic targets and detect the disease earlier. Peripheral blood mononuclear cells (PBMCs) play a role in the deleterious cascade and reflect molecular events associated with PD. Moreover, the study of genetically determined forms of PD enables the identification of subjects at a very early. We hypothesized that PBMCs could be an interesting model to study some mechanisms reflecting the neurodegeneration even at an early stage of the disease. Therefore, we conducted transcriptomic studies in different groups of PD subjects or patients with mutations in order to detect deregulated genes and signaling pathways.We first studied the gene expression profile of PD subjects with the mutation G2019S of the LRRK2 gene. Analysis of microarrays identified disturbances in cell signaling pathways involved in PD. Alterations in the MAPK pathway, the actin cytoskeleton and vesicular transport, associated with the pathogenesis of LRRK2, were noted. The list of deregulated genes separates individuals based on their genetic status including an asymptomatic subject. G2019S LRRK2 mutation is associated to a particular gene expression profile identifiable in PBMCs even at early stage.Then we investigated another form of genetically determined by duplication of SNCA gene. We better characterized the relationship between genotype and clinical phenotype of the subjects. The duplication extends 4.928 Mb, contains 31 genes and results from non-allelic homologous recombination. The analysis of the expression of genes in the PBMCs of a subject carrying the mutation at preclinical stage showed overexpression of SNCA.We compared PBMCs gene expression of G2019S LRRK2 mutation carriers, SNCA duplication carrier and also sporadic PD patients. Our analysis showed that carriers of the LRRK2 mutation and sporadic PD patients have common deregulated signaling pathways that reflect the PD pathogenesis. By contrast, pathways deregulated in the subject with SNCA duplication reflect the pathogenesis of SNCA. In addition, we looked at the expression of SNCA isoforms in PBMCs of these three groups of individuals. Sporadic and LRRK2 patients showed a decreased expression of four isoforms of SNCA in their PBMCs. However, in the duplicated subject, only isoform 112 was overexpressed.Then we used this technology to identify molecular pathways associated with spino-cerebellar ataxia type 2 (SCA2), which provides rarely a parkinsonian phenotype and compared with subjects with a cerebellar phenotype. Again, we identified deregulation of gene expression associated with SCA2 pathogenesis, such as amyotrophic lateral sclerosis and actin cytoskeleton in PBMCs of subjects with parkinsonian and metabolism of RNA and inositol phosphate in cerebellar subjects.Finally, we looked at gene expression in PBMCs according to the evolutionary and clinical stage of PD including individuals at a very early. We compared their gene expression profiles with more advanced PD patients. From the early stages, we observed a deregulation of ERK/MAPK and PI3K/Akt pathways that control cell survival; these findings underscore the importance of these biological pathways in the development of PD.In conclusion, we demonstrated that PBMCs are an interesting model. The transcriptomic studies can get insight into the mechanisms associated with early stages of degeneration and into biological markers, such as SNCA. This technique could be applied in a larger number of subjects including other neurodegenerative diseases to detect specific diagnostic markers of PD.
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Methods to Detect Apoptosis in Equine Peripheral Blood Neutrophils from Normal Healthy Adult HorsesWereszka, Marta 29 August 2007 (has links)
Apoptosis is a form of "planned cell death" and is an essential component of normal tissue differentiation and functional regulation. Neutrophil apoptosis facilitates down regulation of the inflammatory response while minimizing "by stander" injury to normal tissue, and disruption of this process by various diseases may have a significant negative impact on patient recovery. Consequently, neutrophil apoptosis has been the focus of research in many species. However, methods for measuring apoptosis have not been evaluated in the horse. The goal of this study was to adapt previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species for use in equine peripheral blood neutrophils.
To achieve this goal the experiment was divided into three parts: 1. Induce apoptosis and necrosis in equine peripheral blood neutrophils using previously used known inducers and examine the relationship between exposure time and percentage of affected cells; 2. Measure percentage of apoptosis and necrosis using three methods of detection: a) Annexin-V Fitc PI assay, b) Homogenous caspase 3/7 assay and c) Light microscopy and; 3. Compare the results between the three methods of apoptosis detection to determine if results are comparable
The hypothesis was that previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species can be adapted for use in equine peripheral blood neutrophils.
Venous blood samples were collected aseptically from the jugular vein of eight horses. Isolation of neutrophils was performed using density gradient centrifugation on percoll. In part 1 of the experiment aliquots of the neutrophil suspension were cultured in the presence of four known inducers of apoptosis; actinomycin D, staurosporin, cycloheximide and sodium hypochlorite, at four different concentrations (table 2). A fifth population was to induce necrosis using a freeze-thaw cycle and bleach. A control sample was examined (no inducer) to determine spontaneous rate of apoptosis. The aliquots were cultured and the percentage of apoptosis determined at two sequential time points for each horse. Apoptosis was measured at either 30 minutes and 3 hours or 6 and 12 hours by three simultaneous methods: (1) annexin-V FITC PI assay (AVF), (2) homogenous caspase assay (HC) and (3) light microscopy (MS). The AVF and HC methods detect events associated with early apoptosis whilst MS detects nuclear changes which are late events of apoptosis. Using AVF and MS apoptotic cells are able to be differentiated from necrotic cells.
In part two of the experiment the agreement and reproducibility between AVF and MS was further examined. In this part of the experiment neutrophils were isolated from the peripheral blood of 10 normal healthy adult horses. Each isolated sample was cultured with 80µM Actinomycin D for 12 hours and a control sample (no inducer) also prepared. Three triplicate samples were next set up from both the induced and control sample and apoptosis was determined using both AVF and MS.
In part 3 of the experiment, data was analyzed using the mixed model ANOVA following log transformation of the data. Main effects of treatment, concentration and time were analyzed. Statistical significance was considered if P was < 0.05.
The relationship between the three techniques; light microscopy, flow cytometry and the fluorescent plate reader, was investigated using Spearman rank correlation coefficients (Fisher's Z transformation). The Bland-Altman approach for method analysis was used to further characterize the correlation between results obtained via light microscopy and flow cytometry. Statistical significance was considered if P < 0.05.
All inducers increased the percentage of apoptotic cells at either one or more time point and results were most comparable between AVF and MS. Increasing exposure time increased percentage of apoptotic neutrophils for all inducers using AVF and MS (p<0.0001). For both AVF and MS, cycloheximide and staurosporin induced apoptosis significantly above control levels at 3, 6 and 12 hours; actinomycin D at 6 and 12 hours and bleach at 3 and 6 hours as well was 12 hours for AVF only. With HC induction of apoptosis was detected earlier with bleach at 30 minutes and 3 hours and staurosporin at 30 minutes, 3 and 6 hours. Apoptosis was detected only at 6 hours for cycloheximide.
Increasing concentration of inducer significantly increased the percentage apoptotic cells for staurosporin and cycloheximide between the lowest and highest concentration using AVF (p<0.001). For both AVF and MS, increasing concentration of bleach decreased the percentage of apoptotic cells (p<0.05). Increasing the concentration of staurosporin resulted in an increase in apoptosis at 30 minutes and 3 hours.
Both bleach and the freeze-thaw cycle induced necrosis at all time periods excluding 30 minutes for the freeze-thaw cycle (p<0.0001).
Spearman rank correlation coefficients revealed a very high correlation for percentage apoptosis and necrosis between AVF and MS (r2 = 0.91, 95% CI 0.89 – 0.93). A high correlation was also present for AVF and HC (r2 = 0.75, 95% CI 0.69 – 0.79) and MS and HC (r2 = 0.76, 95% CI 0.71 – 0.81). The lower limit of the confidence intervals suggests there is some concern about the similarity between AVF, HC and MS, HC.
The Bland and Altman statistical approach indicates that both AVF and MS are highly reproducible methods with minimal variation between the triplicate samples (AVF: 8.9%, 95% CI 6.25 – 11.6%, MS 7.9%, 95% CI 6 – 9.8%). The mean difference between the two methods is 6.7% (95% CI 3.89 – 9.42%). The 95% limits of agreement indicate that results from MS can be 8.7% below to 22% above results from AVF (95% CI -13.41 – 26.7%).
These findings indicate that caspase activation may occur prior to phosphatidylserine externalization and visible nuclear changes, which is in accordance with previously published data. We discovered that actinomycin D induces significant and reproducible equine peripheral blood neutrophil apoptosis in a time dependant fashion. Similarly, necrosis results from a freeze-thaw cycle or high concentration of bleach and is suitable as a positive control for necrosis. Apoptosis was effectively detected using AVF assay and results indicate good correlation between AVF and MS with an acceptably low mean difference. MS could serve as an inexpensive, simple and quick on site method to rapidly verify results attained from AVF. Induction of apoptosis using the HC was not consistent and can not be recommended based on the results of this study. Future investigation aimed at evaluating assays multiplexed to the AVF which detect other aspects of the apoptotic pathway would lead to increased confidence of results and further evidence of the mode of cell death prior to undertaking clinical studies. / Master of Science
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Investigation of the immunomodulatory properties of intravenous immunoglobulin G (IVIg)Pain, Elisabeth January 2002 (has links)
No description available.
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An Upregulation of DNA-Methyltransferase 1 and 3a Expressed in Telencephalic Gabaergic Neurons of Schizophrenia Patients Is Also Detected in Peripheral Blood LymphocytesZhubi, A., Veldic, M., Puri, N. V., Kadriu, B., Caruncho, H., Loza, I., Sershen, H., Lajtha, A., Smith, R. C., Guidotti, A., Davis, J. M., Costa, E. 01 June 2009 (has links)
Several lines of schizophrenia (SZ) research suggest that a functional downregulation of the prefrontal cortex GABAergic neuronal system is mediated by a promoter hypermethylation, presumably catalyzed by an increase in DNA-methyltransferase-1 (DNMT-1) expression. This promoter hypermethylation may be mediated not only by DNMT-1 but also by an entire family of de novo DNA-methyltransferases, such as DNA-methyltransferase-3a (DNMT-3a) and -3b (DNMT-3b). To verify the existence of an overexpression of DNMT-3a and DNMT-3b in the brain of schizophrenia patients (SZP), we compared their mRNA expression in Brodmann's area 10 (BA10) and in the caudate nucleus and putamen obtained from the Harvard Brain Tissue Resource Center (Belmont, MA) from both nonpsychiatric subjects (NPS) and SZP. Our results demonstrate that DNMT-3a and DNMT-1 are expressed and co-localize in distinct GABAergic neuron populations whereas DNMT-3b mRNA is virtually undetectable. We also found that unlike DNMT-1, which is frequently overexpressed in telencephalic GABAergic neurons of SZP, DNMT-3a mRNA is overexpressed only in layer I and II GABAergic interneurons of BA10. To ascertain whether these DNMT expression differences observed in brain tissue could also be detected in peripheral tissues, we studied whether DNMT-1 and DNMT-3a mRNAs were overexpressed in peripheral blood lymphocytes (PBL) of SZP. Both DNMT-1 and DNMT-3a mRNAs are expressed in the PBL and although DNMT-3a mRNA levels in the PBL are approximately 1/10 of those of DNMT-1, the comparison of the PBL content in NPS and SZP showed a highly significant 2-fold increase of both DNMT-1 and DNMT-3a mRNA in SZP. These changes were unaffected by the dose, the duration, or the type of antipsychotic treatment. The upregulation of DNMT-1 and to a lesser extent that of DNMT-3a mRNA in PBL of SZP supports the concept that this readily available peripheral cell type can express an epigenetic variation of specific biomarkers relevant to SZ morbidity. Hence, PBL studies may become useful to investigate a diagnostic epigenetic marker of SZ morbidity.
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Immaturité, sénescence et hiérarchie fonctionnelle des progéniteurs endothéliaux humains / Immaturity, senescence and functional hierarchy of human endothelial progenitor cellsFerratge, Ségolène 30 September 2016 (has links)
Les ECFC (endothelial colony forming cells) sont considérées comme des cellules endothéliales dérivées des progéniteurs endothéliaux circulants in vitro. Les capacités angiogéniques de ces cellules leur confère un intérêt thérapeutique pour traiter les lésions ischémiques résultant des pathologies cardio-vasculaires. Les ECFC isolées à partir de sang périphérique adulte (ECFC-SPA) sont moins fonctionnelles et perdent leur capacité à former des vaisseaux sanguins persistants contrairement aux ECFC de sang de cordon ombilical (ECFC-SC). Une meilleure connaissance des mécanismes impliqués dans cette dysfonction est requise pour comprendre et prévenir l’altération des ECFC-SPA au cours du vieillissement en vue d’une stratégie autologue. De même, l’utilisation des ECFC-SC en thérapie cellulaire hétérologue est conditionnée par une caractérisation fonctionnelle plus poussée de cette source de cellules angiogéniques.Ce travail de thèse a permis d’établir un profil précis du rendement en ECFC du SPA et du SC. Aucune corrélation avec l’âge du donneur n’a été mise en évidence. Les ECFC-SC sont particulièrement hétérogènes, générant de 0 à plus de 100 colonies. Les échantillons générant moins de 10 colonies présentent une altération fonctionnelle similaire aux ECFC-SPA, associée à une perte de leur caractère immature. Les ECFC-SC les plus clonogéniques sont également les plus angiogéniques. La clonogénicité primaire des ECFC apparait donc comme le marqueur le plus précoce accessible au cours de leur culture permettant de prédire leur future fonctionnalité. Ce pourrait être un critère pertinent pour hiérarchiser et sélectionner les ECFC-SC les plus efficaces en thérapie cellulaire. / ECFCs (endothelial colony forming cells) are derived from endothelial progenitor cells in vitro. Angiogenic capacity of these cells confers a therapeutic benefit for treating ischemic injuries resulting from cardiovascular pathologies. ECFC isolated from peripheral blood adult (AB-ECFC) are less functional and lose their ability to form long lasting blood vessels in contrast to umbilical cord blood ECFC (CB-ECFC). A better understanding of mechanisms involved in this dysfunction is required to understand and prevent alteration of AB-ECFC during aging for autologous strategy. Similarly, the use of CB-ECFC in heterologous cell therapy is conditioned by further functional characterization of this source of angiogenic cells.This thesis has established a specific profile of ECFC yield from adult and cord blood. No correlation with donor age has been demonstrated. The CB-ECFC are particularly heterogeneous, generating from 0 to more than 100 colonies. Samples generating less than 10 colonies have a similar dysfunction than AB-ECFC, associated with loss of their immature features. The most clonogenic CB-ECFC are also the most angiogenic. The initial clonogenicity of ECFC therefore appears to be the earliest marker available during their culture to predict their future functionality. It could be a relevant criterion for classifying and selecting the most effective CB-ECFC cell therapy.
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The Role of Neutrophil Apoptosis in Horses with Acute Abdominal DiseaseKrista, Kathryn Morton 15 June 2012 (has links)
Neutrophils, the chief phagocytic cells in most mammals, are critical in the inflammatory response. Regulation of neutrophil activity occurs through several mechanisms, including apoptosis. Dysfunction of neutrophil apoptosis has been implicated as a cause of organ damage in hyper-inflammatory conditions in human patients. This pilot study investigated apoptosis in circulating neutrophils from horses with surgical lesions in the large and small intestine. We hypothesized that delayed neutrophil apoptosis occurs in peripheral blood of horses undergoing surgery with acute abdominal disease, compared with elective orthopedic cases. Adult horses undergoing surgery for acute abdominal disease (N=10) and elective orthopedic surgery (control) (N=10) were studied. Peripheral blood was collected preoperatively and postoperatively. Neutrophils were isolated using Percoll gradient. Cells undergoing apoptosis were determined by flow cytometry using a commercially available staining kit (Annexin V-PE Apoptosis Detection Kit I, BD Pharmingen™). The Mann-Whitney U test was used to detect significant differences in neutrophil apoptosis between the two groups as well as between lesion types in the abdominal surgery group. Correlations between neutrophils in apoptosis and postoperative parameters were detected using Spearman's rank correlation coefficient. No significant differences in percentages of apoptotic neutrophils between groups were found; however, a significantly lower percentage of neutrophil apoptosis was present in horses with strangulating intestinal lesions versus nonstrangulating lesions. Current investigations about neutrophil apoptosis in human medicine may result in therapeutic intervention to prevent organ damage in hyper-inflammatory states. Understanding the role of neutrophil apoptosis in equine acute abdominal disease may guide the use of new treatments as they become available. / Master of Science
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Genotoxic Effects in Peripheral Blood and Sperm in Humans in Healthy Individuals and Those with Disease StatesAnderson, Diana, Baumgartner, Adolf, Najafzadeh, Mojgan 01 May 2018 (has links)
No / The Comet assay is one of the most versatile tools in toxicology today and can be used to
measure responses in both diploid (peripheral blood lymphocytes) and haploid (sperm)
primary cells in humans. This chapter will discuss how these cells are employed to
determine if they have differential responses to chemical and physical agents in healthy and
disease-affected individuals and how such information can be of use to man.
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Expression Profiling Elucidates a Molecular Gene Signature for Pulmonary Hypertension in SarcoidosisSingla, Sunit, Zhou, Tong, Javaid, Kamran, Abbasi, Taimur, Casanova, Nancy, Zhang, Wei, Ma, Shwu-Fan, Wade, Michael S., Noth, Imre, Sweiss, Nadera J., Garcia, Joe G. N., Machado, Roberto F. 12 1900 (has links)
Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH (n = 8), sarcoidosis patients without PH (n = 17), and healthy controls (n = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.
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